Abstract
Background ; We here present a first intraspecific eco-variant post natal attempt at evaluation of skull signaling in the small African pangolin (Boreoeutheria) family, sub-family phatagininae emphasizing evolutionary trend in ontogeny. Forty; digitally processed skulls and foramen magnum outlines of this species from different geo-locations were assessed for asymmetry, foramen magnum shape variations and modularity hypotheses on skull areas using geometric and Elliptical Fourier analyses methods. Results ; Regression of log transformed centroid size and asymmetric components suggested significant expression of asymmetry through ontogeny (p˂0.03) with size accounting for 81.34% of asymmetric shape changes. Multivariate analysis of regression confirmed directional but low (p˂0.5) fluctuating asymmetry (F1539=3.4045, F882= 3.2665, for dorsal and ventral views respectively). Intraspecific ontogenetic allometric trajectories followed rostro-caudal and caudo-lateral directions; intercepts for shape/size predictions were parallel. Mahalanobis distances between centroids (2.42) of specimens were significant (p˂ 0.01). Variance-covariance matrix in ontogeny lies between 0.0017 and 0.56 suggestive of shape overlaps and variations. Foramen magnum outline descriptors by incremental harmonics explained morphologic details; the first 4 effective principal components defined 96.98% of shape properties, while (3.02%) constituted finer details. 74.1% accuracy decline after size factor elimination. Modulation PCA: of Covariance Matrix and Asymmetry component was 88.38% and 7.48% (PC1 and 2) variance % predicted 10.08%. Conclusions ; The study confirmed directional ‘handedness’ and fluctuating asymmetries among skull samples studied, an attenuated ability to maintain paired symmetric bilaterality irrespective of geo-location. Foramen magnum shape assumed priority over size in ontogeny with profound asymmetry (from the 5th harmonic); an indication of precocity and early presumptive form for arboreal adaptation this observation in conjunction with modularity inference suggested instability and global weakness of cranial integration processes and modules. These findings will be of value for species conservation and may be vital in pangolin evolutionary systematics.
Keywords- African Small Pangolins, Allometry, Foramen magnum, Asymmetry, Skull modularity, Geometric morphometrics
Background
The small African pangolin (SAP) is a poorly described scaly anteater formerly classified as a single family Manidae from the Order Pholidota (Murphy et al., 2001a). Recent research has reclassified those (P. tricuspis ) to the Clades of Boreoeutheria, Sub-order Ferungulata, sub-family Phatagininae (Gaubert et al., 2017; IUCN, 2018) and are not related to the giant anteater (Myrmecophaga tridactyla vermilingua ) of the Order Pilosa from Central South American nativity whose familiarity to sloths is well documented. Pangolins are better related to carnivorans in evolutionary trends (Gaudin and Wible, 1999; Murphy et al., 2001b). All eight extant species of SAP are currently classified as threatened, three of these are critically endangered (IUCN, 2018). SAP are arboreal in nature with Sub-Saharan African geographic distribution (ranging from West, Central to Southern Africa), they are deeply diverged from the Asian and Indian species of the genusManis (Gaubert et al., 2017; Gaudin et al., 2019). Several morphological variations of its head and cranium have brought this species to fore in current researches and further tilted her already threatened status to critical level (Ferreira-Cardoso et al., 2020); this new status justified this study more so that morphological distinction of species is a requisite vulnerability evaluation in the instance of ecology and evolutionary status classification. The species has been declared the most trafficked in the world (Gaubert et al., 2017); this fact serves a major impetus to study its peculiar cranial morphology as well as certain developmental/ biological processes as an adjunct to molecular tracing of trafficked mammals for preservation and conservation. Tracking and tracing smuggled species skeletons and remains through illegal trade routes often require adequate information on geographic distribution, shape and sizes of preserved antiquated or archival specimen as well as season of interception to offer effective control and discourage poaching.
Attempts at preservation and conservation of critically threatened species such as the small African Pangolins (Phataginus tricupis ) have been made through extensive captive breeding; this is partly due to shrinking ecology and urbanization and have been documented to interfere negatively with natural selection (Hewitt, 2001); a phenomenon associated with founder’s events as well as increased occurrence of developmental aberrations in species (Hewitt, 2001), hybridization, is another common occurrence when wild-taken variants are introduced in breeding programs. Preservation effort may only be successful when baseline information on proper characterization and developmental peculiarities of species structural parts is possible (Richtsmeier and Deleon, 2009).
The zygomatic, maxillary and temporal bones of the skull developed from the first pharyngeal arch (Elliot, 2010) whereas the neural crest andmesenchymal cells forms the cranium (Le Lievre, 1980; Le Douarin and Kalcheim, 1999). Modularity hypotheses of development will be tested using landmark partitions to verify if co-variations among embryological related landmarks are lower in comparison to partitions based on anatomical proximities. A combination of three separate embryological compartments will be analyzed for dependency for all partitions in both planes.
Asymmetry as a concept may be defined by the distribution of left-to-right differences in a population (Palmer and Stroebeck, 1986). Fluctuating asymmetry is relevant in the assessment of environmental and developmental stress in organisms. Subtle and unapparent directional asymmetry (DA) has been described fairly regularly among species population using the methods of geometric morphometrics and this seems to be fairly widespread (Klingenberg et al., 1998). The present investigation is to the better of our knowledge a first post natal developmental investigation on the skull of the small African Pangolin. Calvarias development aims at achieving a symmetric paired structure (Elliot, 2010), deviations from this ’target’ morphology could result from individual or population developmental stress/noise (ecologic input) (Urbanova et al., 2014) and can be useful in making inferences about such biological processes since morphological asymmetry may provide information on a variety of biological mechanisms (Polak, 2003). There is lack of documented evidence on the development of the skull in this species, thus making accurate taxonomic classification and discrimination among similar extant species conjectural especially for trafficked Pangolin. The challenge of skeletal remains recovery and failure of formulated conservation policies resulting from wrong identification created a knowledge gap to be bridged by this study. Visual observation of sample remains of various ages revealed minute subtle skull asymmetries despite the absence of masticatory mechanisms prompting such queries to warrant enquiry into the extent and certainty of developmental errors to signify the existence of developmental instability, poor integration and a suspicion of reduced ability to maintain bilaterality in paired symmetric skull structures taken from an environment with ecologic constraints, an observation relatively scarce in arboreal species.
Significant overlap of body structural similarities and divergence between P. tricuspis and all other extant species and betweenP. tetradactyla and P. javanicus respectively have been reported by Ferreira-Cardoso et al. (2020), a situation which provokes a closer investigation on its (SAP) skull characterization.
Latest available literary evidence on extant Pangolins; Manidae (Ferreira-Cardoso et al., 2020) focused on comparative skull shape variations but was not contextual with phenotype as a product of environmental, ecologic and evolutionary trends in the order Pholidota.
The following are the aims and objectives of this study:
  1. This study aims to investigate the association or otherwise between environment/ecology and skull development in SAP to justify observed skull asymmetries.
  2. That the possibility of ‘handedness’ in skull side’s use and development despite absence of teeth for mastication does not exist in the sample population evaluated (null hypothesis), particularly due to paucity of documented evidence of directional asymmetric investigations in the species from different ecological background under study.
  3. Establish and compare variations among sample populations in foramen magnum outline allometry and possible occurrences of malformations such as dorsal and ventral notches among population for an assessment of stability in this structure.
  4. Assess overall evaluated skull developmental pattern among contiguous landmarks (modularity hypothesis) to confirm or otherwise an association in embryonic skull modules compared to related anatomic structures in its integration process using two blocks Partial least squares (PLS), Overall strength of association between blocks: yielded lowest RV coefficients; Permutation test against the null hypothesis of independence with 10,000 rounds of randomization.
. In evaluation of symmetry/asymmetry in skull construction and architecture, all contributory forms of asymmetry (directional asymmetry, fluctuating asymmetry and antisymmetry) were explored (Palmer and Strocker, 1986). We also aimed at studying the foramen magnum construction in ontogeny from the three ecologies as a corroborative attempt in characterization of observed asymmetry of this structure for confirmation of in-born errors. Further, we endeavor an evaluation of ontogenetic morphological features using a range of harmonics in Elliptical Fourier Analysis in resolution of foramen magnum outlines architecture.
0. Materials and Methods
ETHICAL CONSIDERATIONS
Ethical approval for the use of these animals was granted by the Animal Care and Use, Research Ethics Committee (ACUREC), University of Ibadan, Nigeria, ethical code number UIACUREC/17/0023.
ANIMAL ACQUISITION, SKULL PROCESSING AND CATEGORIZATION
To enhance validity, samples for this study were taken from separate locations in Nigeria. All skulls were obtained after due permissions from museums curators of Universities in Nigeria; Eighteen (18), twelve (12) and six (6) from the Department of Anatomy, University of Ilorin, Kwara State, Department of Veterinary Anatomy, Federal University of Agriculture Makurdi, Benue State, and Department of Veterinary Anatomy, University of Ibadan, Oyo State, respectively. Four (4) unsexed were wild taken in Ibadan, Oyo State, categorization was based on skull size and geographical location (Fig. 1) while animal handling was in conformity with ACUREC and NIH guidelines for Use and Care of Laboratory Species. Sacrifice and maceration process were according to Catania et al., (2000) and Igado (2017).
Figure 1 Distribution areas (starred) of the small African Pangolin (Phataginus tricuspis ) along the coast line in the tropical rain forest zone with special concentration in the Southwestern parts of Nigeria which serves as our assessment areas
A series of eleven (11) landmarks comprising of four (4) single midline (axis of symmetry) and seven (7) paired points on either side of the skulls were digitally placed on direct dorsal and ventral views (nine landmarks) of skull images (Fig.2a) using TPS Dig2 Vers. 1.40 (Rholf, 2015) CANON EFS-1200D Camera with EF-S 18-55 IS 11 kit and HAMA tripod plumb line stand stabilizers at a focal distance of 2.8 cm and 15 cm DIN, shutter speed ISO 1/100. The study utilized Generalized Procrustes Analysis (GPA), which superimposed landmark configurations, apart from providing information on size (Centroid size, CS, calcu­lated as the square root of the sum of squared distances of each landmark from the centroid of the landmark configu­ration) and shape (Procrustes distance estimates) eliminates spatial variation that does not correspond to form. Allometric data could therefore be assessed for evaluation as analysis of size and shape differentiation across the geo-ecologies.
Sage: software was used in the evaluation of asymmetric and symmetric components for this study, Symmetry and Asymmetry in Geometric Data Version 1.21 software (Marquez, 2014) freely available online at http://www-personal.umich.edu/~emarquez/morph/